Method of estimating blood volume

ABSTRACT

Disclosed are methods, materials and devices for approximation of blood volume in a fluid, such as in a biological fluid collected during a surgical procedure. The method and devices include the use of a RBC flocculant, such as polyDADMAC, and an approximate blood hematocrit for the type of animal, as well as a calculated RBC packing ratio corresponding to the collection device being used. Also provided is a Blood Indicator Panel (BIP), comprising a series of markings calculated from an observed red blood settlement volume, the average animal type hematocrit, and a calculated RBC packing ratio “η” value for the collection device. Pediatric (about 200 ml or 250 ml size container), adult human (about 1,000 ml-1,500 ml) and veterinary (about 500 ml-2,500 ml) collection containers are also disclosed, that include a RBC flocculant, for use in approximating blood volume in a fluid.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication 62/445,067, filed Jan. 11, 2017, which is incorporatedherein in its entirety.

BACKGROUND OF THE INVENTION

During surgery, blood, saline, and in some cases, small tissue areevacuated from a subject's body, and collected in a collectioncontainer. Typically, this evacuation process is accompanied by suction.The components in the collection canister include at least some volumeof blood in most cases. However, the volume of blood included isdifficult to assess with any degree of accuracy. Blood loss assessmentis critical to the health of the subject, but currently is assessed byonly crude techniques associated with the counting of saline unitsemployed during the surgical procedure by the attending health careprofessional/anesthesiologist. At the conclusion of the surgery,typically the anesthesiologist will estimate the amount of blood lost bydetermining what volume of saline was used during the surgery overall,and then subtracting this saline volume from the total volume of mixedmaterial collected in the collection canister(s).

Blood is composed of RBCs, and RBCs require a significant amount of time(over 3-6 hours) to settle out of a blood/fluid mixture collected at thetime of surgery. Apart from the significant time required to provideeven a reading of settled RBC volume, it has been found that the settledRBC volume does not provide an adequate approximation of blood volumefor assessing blood needs. Current techniques for estimating blood lossare fraught with challenges, including human error (counting “used”saline bags, accounting for residual saline) and serious timeconstraints associated with conventional blood-containing fluidanalysis.

Failure to accurately approximate blood loss during a surgical eventresults in a number of potentially serious health complications to thepatient, as well as increased medical costs. For example,underestimation of blood loss can result in failure to provide a neededblood transfusion to the subject. This in turn results in the subject,in many cases, becoming anemic, requiring extended hospitalized tostabilize the patient and/or even death. An over estimation of bloodloss may result in unnecessary blood transfusions and/or otherunnecessary medical interventions, as well as in waste of blood units.Such may also cause increased risk of disease transmission (for exampleHIV, hepatitis).

The medical arts remain in need of improved techniques and materials toaccurately estimate blood loss. Methods for more accurately monitoringand approximating blood volume in a fluid are needed. Fluid collectionproducts suitable for providing a more accurate blood volumeapproximation are also needed, as will serve to reduce medical costsassociated with unnecessary medical treatments and interventions thatoccur as a result.

SUMMARY OF THE INVENTION

In a general and overall sense, the present invention providesmaterials, devices and methods for more reliably and accuratelyapproximating blood volume in a liquid, especially in a liquid collectedduring a surgical procedure.

Within this disclosure, it should be understood that thecharacterization of red blood cell (RBC) volume separated from a fluidcontaining blood plasma, saline or any other fluid (e.g., urine), doesnot mean an absolute or total separation, but instead an approximationof the stable sedimentation of RBCs in the fluid. RBC sedimentation isto be assessed at room temperature, and relates to the settlement ofRBCs in a sample liquid by gravity (no centrifugation).

Methods of Approximating Blood Volume in a Liquid. In one aspect,methods for measuring blood loss from a mammal, such as a human orveterinary animal, using a RBC flocculant, are provided. The method insome embodiments comprises providing a liquid in a container havingtherein a RBC flocculant, wherein the RBC's within blood contained inthe liquid will sediment to permit a visual and essentially simultaneousRBC sedimentation volume. While settled RBC volume does not equate toblood volume in a liquid, the RBC sedimentation volume is used toapproximate blood volume in the liquid. The method employs a RBCsedimentation volume observed in the presence of a RBC flocculant, theaspect ratio of the collection device and an average hematocrit, toprovide a visually ascertainable approximation of blood volume in theliquid. The visually ascertainable approximation of blood volume in aliquid/fluid containing or possibly containing blood using the presentdevices and methods may be achieved in less than an hour, and as quicklyas within 15-30 minutes, using the techniques described herein. The veryshort time frame facilitated for providing RBC sedimentation in thepresence of an RBC flocculant, and the consequent visual assessment ofapproximate blood volume, provides significant advantages in maintainingthe well-being of the patient, as well as significant resource savingsto the health care provider.

The methods may also be used to detect the presence of blood in aliquid, and in this manner, may be used to test a material (such as foodmaterials, water, pharmaceuticals, etc.) for the presence of bloodcontamination. The material in question would be placed in contact withthe RBC flocculant described herein, and examined for presence of RBCsedimentation. RBC sedimentation would indicate the presence of blood inthe material.

Blood Indicator Panel. In some aspects, a Blood Indicator Panel (BIP)having defined demarcations calibrated to correlate with anapproximation of blood volume in a fluid containing or suspected tocontain blood is provided, as well as methods for preparing a BIPspecific for blood from a particular mammal (human/non-human). Specificcollection devices associated with the defined BIP are also presented.

The BIP may be used in conjunction with any conventional fluidcollection devise and/or collection bag, in the presence of an RBCflocculant, to provide an approximation of blood volume in a fluid. TheBIP employs a calculation that incorporates an estimated averagehematocrit of the blood type being assessed as well as a device packedRBC volume determined for a collected fluid containing blood. A BIP isspecifically provided for veterinary (equine, bovine, canine, feline)human and human uses (adult, infant). Reference to the BIP provides anefficient manner (without need for performing individual calculation)for estimating blood volume in a mixed fluid/liquid by cross-referenceto settled RBC volume on a collection device. In some embodiments, theBIP may be used without conventional volumetric measures, and includeonly the calibrated blood volume markings for identification of bloodvolume in a liquid. In such cases, the type of blood and device has beenpre-calibrated to provide approximated blood volume measures withoutreference to settled RBC levels.

RBC Flocculants and RBC Flocculation in a Fluid/Liquid. In one aspect,particular flocculants are identified that are suitable for flocculatingRBCs, and thereby facilitate the sedimentation of RBCs by gravity.Combined with a biological fluid, such as the aspirate or other surgicalrun off collected during a surgical or other medical procedure episodes,the RBC flocculant will facilitate the rapid sedimentation of RBC'swithin a mixed fluid, such as within about 15 to about 30 minutes,compared to several hours that would otherwise be required for RBCs in afluid to settle in the absence of a RBC flocculant.

As used in the description of the present invention, flocculation isdefined as the coalescence or formation of clusters of RBCs, within afluid comprising blood or potentially comprising blood. The methods maytherefore be used to detect blood contamination, as well as to quantifyan amount of blood in a material by providing for the detection of RBCsin the material and approximately blood volume according to the methodsdisclosed herein.

Fluids potentially comprising blood include saline, surgical aspirate,urine, bile, other biological waste materials, saliva, tissuepreparations, digestive fluids, cerebral fluids, lymph, peritonealfluid, amniotic fluid, and any mixture or combination of these. In thisregard, virtually any moiety or chemical agent that is capable ofpromoting the coalescence of RBCs within less than about 30 minutes atroom temperature to provide a stable settled RBC level, is considereduseful as a RBC flocculant for purposes of providing the present devicesand for use in the present methods. It is envisioned that virtually anymoiety or chemical entity that may impart a positive surface charge to anegatively charged RBC would be useful to promote a more rapidcoalescence, or flocculation, of RBC's together sufficient to enhancethe speed of RBC sedimentation as part of the present devices andmethods.

RBCs present in a fluid containing blood become bound to each other inthe presence of an RBC flocculant, and in this manner form heavierparticles that settle within the container/bottle/tube/collapsible bagor other vessel within which a sample/material is collected. Once theRBCs settle, the volume of the settled RBCs is recorded and employedtogether with a predetermined hematocrit of the mammal (animal/human),and a calculated RBC packing ratio associated with the collectionvessel, so as to provide a real-time estimate of approximate bloodvolume in a liquid or mixed collected specimen.

In some embodiments, the flocculant is polydiallyldimethylammoniumchloride (PolyDADMAC). Virtually any material or chemical may be used asa RBC flocculant in the practice of the present invention, as long asthe material is able to facilitate the aggregation of RBCs to oneanother and decrease the time the RBCs aggregate and form a stablesedimentation level, preferably within about 15 minutes. The amount ofRBC flocculant should be sufficient to facilitate the stablesedimentation of RBCs present in a fluid within less than about 60minutes. In some embodiments, the amount of RBC flocculant should be anamount sufficient to facilitate a stable sedimentation of RBCs in afluid within about 5 minutes, about 10 minutes, about 15 minutes, about20 minutes, about 25 minutes or within about 30 minutes, or within atime range of about 5 minutes to about 20 minutes, at room temperatureand in the absence of centrifugation. The amount of the RBC flocculantshould be sufficient to impart a cationic charge to surfaces of RBCs ina liquid, such as a liquid comprising blood.

Molecules and materials for use as an RBC flocculant include polymericRBC flocculants and non-polymeric RBC flocculants. For polymeric RBCflocculants, the RBC flocculant should have a molecular weight of atleast about 100,000 Da, or about 200,000 Da, 300,000 Da or 400,000 Da,or a mixture of these, and have a relatively positive charge. By way ofexample, the polymeric RBC flocculants may include PEI, PAM, poly(acrylamide-co-acrylate), or any other relatively high molecular weight(greater than or equal to about 1,400,000 Da), positively chargedpolymer capable of imparting a positive charge to the surface of a RBC.Non-polymeric RBC flocculants may include acids, such as HCl or otheracid molecule.

Other suitable high molecular weight cationic polymer RBC flocculantsmay be prepared by polymerization, such as by vinyl additionpolymerization of one or more cationic monomers and by copolymerizationof one or more cationic monomers with one or more cationic monomers.While cationic polymer RBC flocculants may be formed using cationicmonomers, it is also possible to react certain non-ionic vinyl additionpolymers to produce cationically charged polymers. Polymers of this typeinclude those prepared through the reaction of polyacrylamide withdimethylamine and formaldehyde to produce a Mannich derivative.

The RBC flocculant may be used in solid form, in an aqueous solution, asa water-in-oil emulsion, or as a dispersion in water. Representativecationic polymers that may be employed as an RBC flocculant includecopolymers and terpolymers of (meth) acrylamide with dimethylaminoethylmethacrylate (DMAEM); dimethylaminoethyl acrylate (DMAEA);diethylaminoethyl acrylate (DEAEA); diethylaminoethyl methacrylate(DEAEM); or their quaternary ammonium forms made with dimethyl sulfate,methyl chloride, or benzyl chloride. In alternative embodiments, the RBCflocculant may comprise dimethylaminoethylacrylate methyl chloridequaternary salt-acrylamide copolymers, sodium acrylate-acrylamidecopolymers and hydrolyzed polyacrylamide polymers.

After RBCs are settled within a collection device or in a collapsiblebag designed to conform to the dimensions of a collection deviceincluding an RBC flocculant, a settled RBC volume can be estimated usinggraduated markings on the collection device and/or collapsible bag thathave been calibrated to provide a blood volume approximation. A packingratio η may be determined, which is calculated using the settled RBCvolume verses the RBC volume after centrifugation. With thedetermination of packing ratio, a total blood volume, such as a bloodloss volume (V_(b)) or total amount of blood in a collected fluid, inthe collection device can be estimated with the settled RBC volume(V_(m)), the subject's hematocrit (Hct) value (or an average HCTdetermined from a number of similar subjects), and the packing ratio ηaccording to the following formula:

V _(b) =V _(m)/(Hct×η)  1)

Normally, in the absence of a flocculant, the RBC sedimentation rate, orerythrocyte sedimentation rate (ESR) is relatively low, or notdetectable to the unaided eye (See FIG. 18). It may take many hours oreven days for RBCs to settle within a collection container withoutcentrifugation at room temperature in the absence of a flocculant. Whilenot intending to be limited to any particular mechanism of action, thismay be due, at least in part, to the electrostatic repulsive forces thatnaturally exist between RBCs, as RBCs are naturally negatively charged.In the presence of an RBC flocculant, such as the RBC flocculantpolyDADMAC, the RBC settlement rate increases significantly. In thepresence of an RBC flocculant, RBC sedimentation occurred in less thanabout 20 minutes, and formed a stable RBC sedimentation volume that wasreadily visible to the unassisted eye. The RBC sedimentation volume wasconsidered sufficiently stable for purposes of the present methods whenan observable change in RBC sedimentation volume was less than about0.5% per minute.

Canisters, Collection Bags and Containers: Collection devices includingcanisters, collection bags and containers suitable for the collectionand visual estimation of blood loss are provided. The collection devicesmay take the form of a round, square, octagonal, cylindrical, conical orvirtually any configuration. The collection device should besufficiently transparent or at least opaque so as to permit the visualdetection of a level of material, such as sedimented RBCs, within thecollection device. The collection device can be of a solid or flexiblematerial, such as a hard plastic or glass, or a plastic material. Thecollection device may include a series of volumetric demarcationsassociated with the volume of the device itself, and an amount of an RBCflocculant suitable for facilitating RBC flocculation. The RBCflocculant may also be described as having a molecular weight sufficientto permit the settling of RBCs associated with the flocculant withoutcentrifugation and at room temperature, within less than 30 minutes, orwithin about 5 minutes, about 10 minutes, about 15 minutes, or evenabout 20 minutes. The RBC flocculant may be provided in the collectiondevice as a dry weight amount of flocculent, or as an amount offlocculent in a carrier solution, or as a pre-coating on the collectiondevice (such as evenly coated on at least one surface, the entiresurface, on a partial area, or as a strip along the inside of thedevice). These descriptions are provided by way of example and are notintended to provide limitation of potential embodiments envisioned foruse and practice of the present devices and methods.

The following list presents an exemplary description of the treatedand/or flocculant containing collection containers presented:

1,200 ml container (having a canister configuration), 5 ml and/or 10 mlgraduated markings, one or more inlet ports, and one or more outletports (suitable for attachment of a tube suitable for imparting asuction so as to draw a fluid into the receptacle through a tube at thefirst inlet port).

500 ml container (having a canister configuration), 1 ml and/or 5 mlgraduated markings, one or more inlet ports, and one or more outletports (suitable for attachment of a tube suitable for imparting asuction so as to draw a bio fluid into the receptacle through a tube atthe first inlet port).

100 ml, 250 ml or 500 ml container (having a canister or conicalconfiguration), 1 ml and/or 5 ml graduated markings), one or more inletports, one or more outlet ports, where at least one inlet port issuitable for attaching a tube for applying suction and drawing fluidsand other materials into the container. These devices are particularlyapplicable for pediatric uses and smaller surgical procedures (sinussurgeries).

500 ml collapsible envelope (having 1 ml and/or 5 ml graduated markings,1 or 2 inlet ports (resalable), and one or more outlet ports suitablefor attachment of a tube suitable for imparting a suction to draw afluid into the receptacle through a tube at the first inlet port).

100 ml, 200 ml, 250 ml, 300 ml, 400 ml, 500 ml, 1,200 ml, and 2,500 mlvolume collapsible envelope (optionally having 5 ml and/or 10 mlgraduated markings), 1 or 2 inlet ports (resealable) and one or moreoutlet ports (suitable for attachment of a tube for connecting the bagto a vacuum device, so as to draw a fluid into the receptacle through atube at the first inlet port) (FIG. 13). The collapsible bag should beprepared from a plastic material, similar to that of a saline bagcontainer, and will contain a suitable amount of a flocculant, such aspolyDADMAC. The collapsible bag will also include a handle or other loop(FIG. 13, #10), located at one end to facilitate the hanging of the bagonto a mounting, such as an i.v. pole. A BIP may also be included on thebag, or may be placed as an attachment to the bag, which will provide avisually identifiable approximated measure of the volume of bloodcontained in a collected fluid. The bag may also include conventionalvolumetric markings, as are typical for saline and feeding bags.

100 ml, 250 ml and 500 ml conical collection receptacle (optionallyhaving 1 ml, 5 ml, and/or 10 ml graduated markings), an RBC flocculant,1 or 2 inlet ports (resealable) and one or more outlet ports (suitablefor attachment of a tube suitable for imparting a suction so as to drawa fluid into the receptacle through a tube at the first inlet port).These smaller collection devices have particular applications andprovide greater accuracy for smaller (less than about 500 ml) bloodvolume monitoring. These smaller devices, and/or collapsible bagsdesigned to fit within them that include calibrated blood volumemarkings specific for the conical receptacle size, are useful especiallyin neonatal, pediatric and other critical surgical situations.

Method of Estimating Blood Volume and Blood Loss in a Fluid: In anotheraspect a method of estimating blood volume in a liquid is provided. Inparticular embodiments, the method is used to estimate blood volume lossfrom a patient from fluid collected during a surgical procedure.

The method includes collecting a volume of liquid into a collectiondevice that includes a RBC flocculant suitable for imparting arelatively positive charge to the surface of a RBC, for a period of timesufficient to permit RBC sedimentation by gravity within the collectiondevice. The volume of sedimented RBCs after a sufficient period of time,together with an estimated hematocrit of the type of animal/human bloodin the fluid, is used together with a calculated RBC packing ratiodetermined for the particular collection device, to calculate anapproximate blood volume value in the liquid collected, using Equation 1above. Alternatively, these calculations are provided in a calibratedmarking panel called a Blood Indicator Panel (BIP), as part of thepresent invention, which may be included on a blood collection device.As such, an immediate and visual blood volume assessment may be providedas RBCs in fluid collected in the flocculant containing collectiondevice sediments. In some embodiments, the BIP will include a calibratedmarker coinciding to an estimated blood volume of 50 ml, 100 ml, 200 ml,400 ml, and 600 ml, and a volumetric alignment marking for aligning theBIP within a conventionally volumetrically marked fluid collectiondevice (every 5 ml, 10 ml, for example), such as is common on aconventional 1200 ml canister.

Blood Volume Assessment System: Fluid collection Canister and/orEnvelope and Tubing System: The invention also provides a kit that willinclude any one or more of the above receptacles including anappropriate amount of an RBC flocculant and calibrated with blood volumedemarcations, a first length of tubing suitable for aspirating a fluidfrom an area into a first port on the receptacle, a second length oftubing suitable for connecting with a source of suction and a secondport of the receptacle, so as to impart a vacuum in the receptacle. Thesystem may be provided as a kit, and may also include an instructionalinsert.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. 50 ml of 20%, 40%, and 65% bovine blood mixed with 200 ul of a3× diluted Superfloc C-591 stock solution. 1—collection canister,2—level of settled RBCs (by gravity), 3—level of settled RBCs (bygravity), 4—level of settled RBCs (by gravity), 6—total fluid level incanister, 7—total level in canister, 10—flocculant.

FIG. 2. Containers with different aspect ratios have different red bloodsedimentation rates. 1—graduated conical tube, 2—graduations at 0.5 mlintervals on conical tube, 3—level of sedimented RBCs (gravity), 4—100ml collection canister, 5—total fluid level in canister, 6—level ofsedimented RBCs (gravity), 10—flocculant.

FIG. 3A-3D. FIG. 3A—Settlement Volume Change with Time (30% blood insaline) the change in RBC sedimentation volume over time forblood/saline mixture sample with 30% blood concentration. FIG.3B—Settlement Volume Change with Time (40% blood in saline). The changein red blood sedimentation volume over time for blood/saline mixturesample with 40% blood concentration. FIG. 3C—Settlement Volume Changewith Time (65% blood in saline). The change in RBC sedimentation volumeover time for blood/saline mixture sample with 65% blood concentration.FIG. 3D—The set-up of the experiment to measure the average RBCsedimentation volume over time. 1—Glass graduated cylinder, 2—sedimentedRBCs (gravity), 2—total fluid level.

FIG. 4. The RBCs in 10 ml of 80% blood sedimented when diluted to53.33%, 40% and 26.7%, respectively, with saline, going from left toright. All concentrations settled to the same packed RBC volume. 1—1200ml collection canister with flocculant, 2—settled RBC level, 3—totalfluid in canister.

FIG. 5A-5B. 5A—Operating room with a collection canister (8) having aflocculant and a cap (15) on an i.v. pole (6), the cap (15) having anaspiration port (2), an inlet port (4) and a vacuum port (11). The inletport (4) may be used to add saline or other diluent to the collectioncanister as needed. The collection canister (8) may include a bloodindicator panel (BIP) (5). The aspiration port (2) will receive a tube(1) that is used to aspirate biological fluids away from an operatingfield during a surgical procedure of a patient (7). 5B—The collectioncanister (8) will include a cap (15) having an aspiration port (2), aninlet port (4) and a vacuum port (11). Aspirated fluids from a patientwill be evacuated into the collection canister (8) by way of a suctionbeing pulled on the collection canister through the vacuum port (11).

FIG. 6. A flocculant as a particle suspension (4) may be applied to theinside walls of a container by ultrasonic atomization (3, 4, 5) of theflocculant material in a carrier solution. Any conventional ultrasonicatomization device (2) and syringe pump may be used to coat a collectiondevice, such as a 1200 ml canister with a flocculant, for example,polyDADMAC.

FIG. 7A. Application of a polyDADMAC coating (flocculant particles (10))onto the surfaces (2) of a collection container (1), such as a 1200 mlcanister using an ultrasonic atomization device (3). FIG. 7B. Thetreated 1200 ml canister (1) with two batches of polyDADMAC coating (3)on the surfaces (flocculant particles, 10).

FIG. 8. Flocculant (10) treated canister having a cap (1), the cap (1)having a first inlet port (4) connecting the canister by a length oftubing (4) to a container having a volume of a blood mixture, and asecond vacuum port (5) that connects the canister through a length oftubing to a vacuum source, so as to create a suction force within thecanister.

FIG. 9A-9D. The change of RBC settlement volume along the 20-minute timein the case of 20% blood/saline mixture experiment. The totalblood/saline mixture volumes are 200 ml (FIG. 9A), 400 ml (FIG. 9B), 800ml (FIG. 9C), and 1200 ml (FIG. 9D), respectively. Markers were createdas part of a BIP to provide an approximation of blood volume (notsettled RBC volume).

FIG. 10A-10D. The change of RBC settlement volume along a 20-minute timeperiod with 40% blood/saline. The total blood/saline mixture volumesare: FIG. 10A—200 ml; FIG. 10B—400 ml; FIG. 10C—800 ml; and FIG.10D—1200 ml, respectively. (Note: the volume measurements on each chartare obtained from the markers prepared for the 1200 ml canister treatedwith flocculant. Markers as part of a blood indicator panel (BIP) wereprovided to express an approximation of blood volume. It is estimatedthat the markers provide an approximation of blood volume (not settledRBC volume), that may vary as much as about 20 ml.).

FIG. 11 A. A stable settlement of RBCs was observed at 10-15 minutes inthe 20% bovine blood/saline mixture in the polyDADMAC (flocculantparticles, 10) coated 1200 ml canister (1). The settled RBC volume inthe canister was observed at a volume level of about 110 ml. (2), and atotal volume of 950 ml (3) according to the volumetric graduations onthe canister. FIG. 11B—A stable settlement of RBCs after about 15-20minutes in the 40% bovine blood/saline mixture in the polyDADMAC(flocculant particles, 10) coated 1200 ml canister (1). The settled RBCvolume in the canister was observed at a volume level of about 240 ml.(2) and a total volume of about 1150 ml. (3), according to theconventional volumetric graduations on the canister.

FIG. 12. Three flocculant-treated canisters (left to right, labeled asI, II, and III), were prepared to include a Blood Indicator Panel (4).As illustrated, the Blood Indicator Panel (4) is presented as a separateindicator panel, and may be provided alongside conventional volumetricmarkings on the canister. The Blood Indicator Panel includes a series ofcalibrated markings that provide an approximation of blood volume. Theblood/saline mixtures examined in each canister were 50% blood (I), 30%blood (II) and 20% (III). The BIP (see vertical white bar), at canister12III shows a settled RBC level of about 50 ml in the presence of aflocculant (flocculant particles, 12), which corresponded to acalibrated BIP reading of about 116 ml.

FIG. 13. Collapsible plastic bag blood receptacle with flocculant,having a BIP (20), an inlet port (3), a vacuum port (4) to be placed onthe container, and a second inlet port (15) to permit addition of salineor other diluent. The bag should also include a hook (10) fashioned topermit the bag to be placed on an intravenous pole or other mounting.

FIG. 14. Conical collection device with flocculant (Critical volume andPediatric applications), having a BIP (5), RBC flocculant (4), an inletport (2), a vacuum port (6) and a second inlet port (3).

FIG. 15. BIP. A typical BIP suitable for use with a 1200 ml collectioncontainer may include a marker for at least the following calibratedapproximate blood volumes: 50 ml, 50 ml, 100 ml, 200 ml, 400 ml, and 600ml. The BIP will provide an accurate approximation of blood volumewithin a collected fluid containing up to about 50% or less blood. TheBIP (20) includes calibrated series of blood volume markings 6 (50 ml),7 (100 ml), 8 (200 ml), 9 (400 ml), and 10 (600 ml). This BPI is shownalongside a volumetric Settled RBC Volume reference tool (1), thatincludes conventional volumetric measurements of a collection device 15(about 25 ml), 4 (about 50 ml), 3 (about 125 ml), and 2 (about 250 ml).The volume of settled RBCs does not provide a direct measure of thevolume of blood (indicated on the BIP) in a collected fluid. Instead,the settled RBC level is a reference point from which an appropriateblood volume may be visually identified.

FIG. 16A-16B. Charted data of rate of RBC sedimentation in control (notheat-treated flocculant coated canisters) and experimental (heat-treatedflocculant coated) canisters. The tests were performed using 1000 ml ofa 20% blood with saline mixture (16A), and 500 ml of a 40% blood withsaline mixture (16B). The heat treated canisters maintained the abilityto flocculate the RBCs in both of the 20% and the 40% blood/salinemixtures introduced into the canisters within 3 minutes (FIG. 16A) andwithin 6 minutes (FIG. 16B) of coming in contact with the flocculantcontaining canister.

FIG. 17A-FIG. 17B. Settlement of RBCs from bovine blood in a hightemperature aged flocculant coated canister (FIG. 17A, Panel I (20%blood), FIG. 17B, Panel I (40% blood) compared to non-high temperaturetreated flocculant canisters (FIG. 17A, Panel II (20% blood), FIG. 17B,Panel II (40% blood).

FIG. 18. Equine Blood Study: Comparison of RBC sedimentation in 100 mlcontainer with or without flocculant (volumetric readings approximatedfrom visual inspection using course volumetric markings on containers.RBC sedimentation in 100 ml container with flocculant. 10% blood withflocculant (w/F); 20% blood with flocculant (w/F); 30% blood withflocculant (w/F); 40% blood with flocculant (w/F); 50% blood withflocculant (w/F); 65% blood with flocculant (w/F); No visuallydetectable sedimentation of RBCs was observed in the 10% blood withoutflocculant (w/o F); 20% blood without flocculant (w/o F), 30% bloodwithout flocculant (w/o F); 40% blood without flocculant (w/o F) (notshown); 50% blood without flocculant (w/o F, not shown); or 65% bloodwithout flocculant (w/o F) mixtures examined at room temperature overthe test period (0 to 25 minutes).

FIG. 19—Equine Fresh Blood Study: RBC sedimentation from a 61.6%blood/saline mixture in a flocculant coated 1200 ml canister. About 400ml of fresh equine blood was combined with 250 ml saline. The volume ofRBC sedimentation was recorded. The RBCs began to settle out within 1minute (500 ml RBC sedimentation volume). The RBCs settled to a volumeof about 300 ml by 15 minutes, and remained stable at this sedimentationvolume until the end of the observation period.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the disclosure pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the present technology, the preferred methods andmaterials are described herein.

As used here, the term “flocculant” is intended to mean a molecule thathas a cationic charge that is capable of facilitating the coalescence ofRBCs in a fluid at room temperature, and form a settled RBC mass withless than 30 minutes at room temperature without centrifugation.

Reference to an element by the indefinite article “a” or “an” does notexclude the possibility that more than one element is present, unlessthe context clearly requires that there be one and only one element. Theindefinite article “a” or “an” thus usually means “at least one.”

As used herein, “patient” or “subject” means an individual havingsymptoms of, or at risk for, cancer or other malignancy. A patient maybe human or non-human and may include, for example, animal such a horse,dog, cow, pig or other animal. Likewise, a patient or subject mayinclude a human patient including adults or juveniles (e.g., children).Moreover, a patient or subject may mean any living organism, preferablya mammal (e.g., human or non-human) from whom a blood volume is desiredto be determined and/or monitored from the administration ofcompositions contemplated herein.

As used herein, “about” means within a statistically meaningful range ofa value or values such as a stated concentration, length, molecularweight, pH, sequence identity, timeframe, temperature or volume. Such avalue or range can be within an order of magnitude, typically within20%, more typically within 10%, and even more typically within 5% of agiven value or range. The allowable variation encompassed by “about”will depend upon the particular system under study, and can be readilyappreciated by one of skill in the art.

The following examples are presented to demonstrate preferredembodiments of the invention.

Example 1—RBC Flocculants

The present example presents materials that may be used as RBCflocculants in fluid containing or suspected to contain blood. Thefollowing chemicals in Table 1 may be provided as RBC flocculants.

TABLE 1 Chemicals Descriptions Gelatin A solution of electrostaticcharged poly-peptides having a wide range of molecular weight.Literature indicates gelatin is able to increase RBC aggregation afteradsorption on a RBC surface. Dextran 80 + CaCl₂ Literature indicatesthat Dextran 80 plus a divalent cation like Ca²⁺ Mg²⁺, and Ba²⁺ willincrease aggregation of RBCs Acid Treatment The isoelectric point ofblood is at pH = 4.75-5. The pH change of blood samples to less than4.75 will convert RBCs from negative to positive surface charge.Polyethylenimine (PEI) with PEI is a polymer composed of large number ofpositively charged different molecular weight amine groups, which isexpected to attract RBCs to cause RBC settlement. Polyacrylamide (PAM)with PAM is widely used as a flocculants for water treatment. It candifferent molecular weight be configured to either positive or negativecharge. Positively charged PAM is toxic to aquatic wildlife. Aluminumsulfate (Alum) Alum is positively charged at neutral pH.Polydiallyldimethylammonium PolyDADMAC is a positively chargedwater-soluble polymer chloride (PolyDADMAC) with different molecularweight

Lab Test and Down Selection:

All the identified chemicals listed in Table 1 were tested and evaluatedfor suitability. Bovine whole blood purchased from Innovative Research(Novi, Mich.) was used to determine the suitability of each chemicalabove as a RBC flocculant. The whole blood contained with sodium citrateas the anticoagulant. The whole blood was diluted with saline to providethe following blood concentrations: 20%, 30%, 40%, 50%, 65%, and 80%,which were examined in the present studies.

Gelatin induced RBC aggregation but did not cause sedimentation. At theend, gelatin converted the entire blood/saline mixture samples into agel structure. Without separating RBCs from the plasma and saline,gelatin is not suitable for this application.

Dextran 80+CaCl₂ also converted the blood/saline mixture samples intogels instead of separating the RBCs from the blood plasma and saline.

Acid treatments of the blood/saline mixture samples were performed byadding 1.8% of high concentration acid 6N HCL. The pH of the samples waschanged to about 4.5. The acid treatment caused aggregation andsedimentation of the RBCs. However, using relatively large amount ofstrong acid is not cost-effective for this application and it isdifficult to handle strong acid in the process. Thus, acid treatment maybe used to increase ESR.

PEI, a positively charged polymer, caused RBC sedimentation from theblood plasma and saline. The PEI must be dissolved under an acidiccondition (i.e., with pH<7.0). In other words, a blood/saline mixturesample at neutral pH has to first have acid added to lower the pH beforePEI can be used to facilitate RBC sedimentation in a biological fluidcontaining blood. The relative effectiveness of PEI and polyDADMAC werecompared. The ESR (erythrocyte settling) induced by PEI was found to belower than that of polyDADMAC. PEI is an acceptable flocculant for RBCsin a blood containing biological material (fluid).

PAM is a polymer that can be either negatively or positively charged.The negatively charged polymer will not induce the negatively chargedRBCs to aggregate and settle. The positively charged PAM may be expectedto be used for this application.

Alum is a positively charged molecule. It did facilitate a lower ESR,but at a much lower rate and amount. The relative molecular weight of aflocculant is considered for use as an appropriate flocculant because itenhances settlement of RBCs from gravity. In the studies with PEI andpolyDADMAC, it was found that the higher molecular weight of thosepolymers caused faster RBC sedimentation.

PolyDADMAC is another positively charged polymer. It is also watersoluble at neutral pH. PolyDADMAC in the presence of blood/salinemixture samples has the ability to rapidly cause RBC sedimentation. Onlya small amount by weight of polyDADMAC is needed to induce thesedimentation. Quantitative studies are described below to determine thefraction of polyDADMAC needed proportionally to the amount of blood in abiological mixture containing blood. These studies also suggested thattoo great an amount of polyDADMAC may hinder, or even stop, RBCsedimentation. This may be because the RBCs become coated and surroundedby the positively charged polyDADMAC, and as such, the coated RBCs repeleach other, by positive charges instead of negative charges, therebyhindering and/or preventing aggregation and sedimentation.

The low-cost, high molecular weight (at least 100 KDa, 200 KDa, 300 KDaor about 400 KDa), positively charged polyDADMAC was identified as apreferred, cost-effective flocculant to quickly induce RBCsedimentation. Other high molecular weight, positively charged polymerflocculants (e.g., PEI, PAM, etc.) would also be useful in the practiceof the present methods and creation of container vessels. It is expectedthat an acid treatment would also be useful as part of the method toprovide RBC sedimentation.

Example 2—Blood Loss Estimation

The present example demonstrates the material polyDADMAC as an effectiveflocculant for RBCs contained in a fluid, and the use of this flocculantfor estimating total blood volume in a fluid that contains blood andother liquids, including saline.

High-molecular weight (at least 100, 200, 300 or 400 KDa) research-gradepolyDADMAC was obtained from Sigma-Aldrich (Catalog number 409022 or409030) along with industrial-grade polyDADMAC. The industrial-gradeproduct is commercially available from Kemira Chemical under the productname “Superfloc C-591”, which contains a 20% concentration ofpolyDADMAC. This material is used in water treatment. Studies wereperformed to compare the effectiveness between the research-grade andindustrial-grade products. The studies revealed that the Superfloc C-591produced similar results in RBC sedimentation as the research-gradeproduct. The cost of the industrial-grade product is lower than that ofthe research-grade product. The purity of C-591 varied, resulting insome differences in RBC flocculation. In the use of research grade andindustrial grade polyDADMAC, the 20 wt. % in water stock solutions werediluted with water to 6.67 wt. % working solution to reduce theviscosity of the solution and to enhance ease of handling. The dryweight of flocculant for a 1200 ml collection canister was about 600 mg.of polyDADMAC.

Amount of Flocculant Needed to Facilitate RBC Sedimentation. SuperflocC-591 (obtained from the manufacturer (viscous liquid, 20 wt. % in H₂O)was used as a RBC flocculant to test a bovine blood and saline mixture.The amount of flocculant needed to facilitate a more rapid RBCsedimentation rate was examined. It was discovered only about 0.4% (v/v%) of the Superfloc C-591 working solution added to the totalblood/saline mixture volume was needed to provide an acceptably rapid(within 15 minutes) sedimentation of RBCs in the mixture. For example,in a 1-liter blood/saline mixture sample, only 4 ml of the SuperflocC-591 working solution was needed, regardless of the blood concentrationin the mixture. This translates to about 320 mg of polyDADMAC flocculantfor a 1.2 liter canister.

FIG. 1 illustrates three 50 ml-volume blood/saline mixture samples withthe blood concentrations (from left to right in the figure) of 20%, 40%,and 65%, respectively. The addition of the same amount of 200 μl (or0.4% of 50 ml) of the Superfloc C-591 working solution induced red bloodsedimentation at all of these blood concentrations. However, thesedimentation rate was found to be different depending on theconcentration of blood in the particular mixture, as discussed below.

Tests also revealed lower or higher concentrations of the SuperflocC-591 working solution may be used. For example, concentrations as lowas 0.3% (v/v %) and as high as 0.6% (v/v %) were found effective,however, the exact upper and lower limits were not determined. It wasfound that a 6% (v/v %) concentration of the Superfloc C-591 workingsolution added to blood/saline mixture sample with a 20% bloodconcentration induced minimal settlement, but that may not be the upperlimit for sedimentation.

Aspect Ratio of Blood Container (Receptacle or Canister). During theexperiments, it was found that the RBC sedimentation rate depends, inpart, on the aspect ratio D:H of the container, where D is the diameterand His the height of the container as shown, for example, in FIG. 2.The aspect ratio D:H depends on the shape of the container. Thesedimentation rate is higher when D:H is higher because a larger Dprovides a larger area or space for the aggregated RBCs to settle bygravity. Therefore, the sedimentation rate will be different whendifferent blood collection containers (or canisters) are used.

Another finding during the experiments is that the Superfloc C-591flocculant sedimented RBCs if it is placed in the container beforeadding the blood/saline mixture samples as well as when added to thecontainer after it is already filled with the blood/saline mixturesamples.

Sedimentation Rate and Blood Concentrations. The present studiesindicated that the blood concentration in the blood/saline mixture alsoaffects the RBC sedimentation rates, with a faster sedimentation rate insamples having a lower concentration of blood versus samples containinga higher concentration. FIGS. 3A, 3B, and 3C, for example, illustratethe sedimentation rates of the RBCs in the 30%, 40%, and 65%blood/saline mixture samples, respectively.

A graduated glass cylinder (FIG. 3D) was used in these studies todetermine difference in the sedimentation rates. Three 60 mlblood/saline mixture samples, with blood concentrations of 30%, 40%, and65%, were used in the experiments. A drop of 240 μL (or 0.4% of 60 ml)of the Superfloc C-591 working solution was added to the glass cylinderfirst. Then a 60 ml blood/saline mixture sample was added to the glasscylinder. The separation of the RBCs was visually observed by a cleardifferentiation of fluid layers within the glass cylinder, with thesettling RBCs being the more opaque fluid layer toward the bottom of thecylinder and the blood plasma/saline layer being the clearer fluid layerabove the settling RBC layer. The settled RBC volume within the cylinderwas recorded by reference to the cylinder graduated markings andrecorded every minute.

The settling RBC volume was set to 60 ml at time 0 since no visualseparation had started at time 0. After time 0, the RBCs begin settlingfrom the blood plasma/saline layer and initially settle toward thebottom of the container at a high sedimentation rate, but thesedimentation rate decreases over time. In this regard, as the amount ofRBCs separated from the blood plasma/saline layer increases there arefewer RBCs to separate from the blood plasma and saline. Eventually, theRBC sedimentation rate is very low, so that the rate of RBC volumechange is less than 0.5% per minute. At this point, the volume ofsettled RBCs is considered stable. In other words, a stablesedimentation is achieved when the volume change of the settled RBCs isless than 0.5% per minute. The “packed RBC volume,” which is denoted asV_(m), is the volume of the RBC layer at the bottom of the containerwhen the RBC sedimentation is considered stable.

FIG. 3A illustrates that the blood/saline sample mixture with 30% bloodconcentration started RBC sedimentation almost instantly after mixingwith the flocculant. Most of the sedimentation was complete within oneminute. A stable RBC sedimentation was achieved around 10 minutes afterthe sample is mixed with the flocculant. The stable packed RBC volumeV_(m) was about 9.5 ml. For the blood/saline mixture sample with 40%blood concentration, the sedimentation rate slowed slightly (FIG. 3B).The most sedimentation of the majority of RBC's occurred at two minutesinstead of the one minute observed for the 30% blood concentrationsample. A stable sedimentation for the 40% concentration sample wasachieved at about fifteen minutes and the V_(m) is about 14 ml. For the65% blood concentration sample, the sedimentation was slower. (FIG. 3C).It took over 60 minutes before a stable sedimentation was observed. Themethods for determining blood volume in a blood/saline mixture wereoptimal and consistent in liquids containing 50% or less blood. Rapidsedimentation is characterized as achieving a stable sedimentation ofRBCs in a saline/blood mixture within fifteen minutes of addition of theflocculant at room temperature (no agitation of the mixture duringsettlement).

To ensure that the blood concentration in a fluid remains below about50%, blood (for optimal RBC separation), additional saline or otherappropriate blood diluent may be added. It was found that blood/salinemixtures with high concentrations of blood can still achieve a rapidsedimentation. One way in which sedimentation may be reinitiated shouldthe sedimentation rate appear to be slowing down, one may add anadditional volume of saline so as to further dilute the blood. Three 10ml blood/saline mixture samples with 80% blood (bovine blood containingsodium citrate anti-coagulant) concentration were prepared in threebottles, as shown in FIG. 4. All samples were mixed with 30 μL (about0.3% of 10 ml) of the Superfloc C-591 working solution (about 2 mg dryweight Superfloc C-591). No sedimentation was observed before thesamples were diluted. Each bottle had saline added to dilute the bloodconcentration and, going from left to right in FIG. 4, 5 ml, 10 ml, and20 ml of saline was added, which diluted the samples to 53.3%, 40%, and26.7%, concentrations of blood, respectively. The container was agitatedslightly to mix the added saline with the sample. Upon dilution, theRBCs began to sediment.

The 26.7% blood concentration mixture and the 40% blood concentrationmixture achieved a stable sedimentation at around 10 minutes. The 53.3%blood concentration mixture achieved a stable sedimentation at about 16minutes. As shown in FIG. 4, the final packed RBC volume V_(m) (i.e.,the darker bottom layer at the bottom of the container) in all threesamples is the same, even in the presence of different amounts ofsaline. This result was achieved because the initial 10 ml of 80% bloodconcentration mixture contains the same amount of blood volume, 8 ml.This study indicated that 1) more saline can be added to dilute ablood/saline mixture to help increase the RBC sedimentation rate and 2)the total blood volume can be estimated through the sedimentation of theRBCs, since the volume of RBCs aggregated at the bottom of the container(i.e., the packed RBC volume) is not affected by the amount of saline ina blood/saline mixture.

It should be noted that with other flocculants or other chemicaltreatments discussed above, further testing using the testing protocolpreviously outlined may be followed to determine optimal flocculantconcentrations and blood/saline ratios.

Repeatability. The RBC sedimentation study using the 40% blood (bovineblood with sodium citrate anticoagulant) concentration mixture (FIG.10A) was repeated three times. Standard deviation error bars are shownat various points on the line in the FIG. 10A plot. The repeatability ofthe experiments was very good, especially at the points on the line whenthe sedimentation approaches stability. Relatively high variationsoccurred initially as shown by the longer standard deviation error barsat those points on the line, but these variations are probably due tovisual reading errors caused by the rapidly changing volume of thesettling RBC layer at those points. The data variation is very smallwhen the sedimentation approaches stability. In other words, thevariation of the packed RBC volume V_(m) is small when the sedimentationis stable.

Total Blood Volume Estimation. To demonstrate the feasibility ofestimating the total blood volume, an algorithm was developed toestimate the total blood volume in a blood/saline mixture based on thesedimented RBC volume V_(m). If the actual RBC volume is denoted asV_(c) (as determined through centrifugation), the total blood volume isdenoted as V_(b), and the patient's hematocrit is denoted as Hct, thenthe following relationships are created:

η=V _(m) /V _(c) , Hct=V _(c) /V _(b) →V _(b) =V _(m)/(Hct×η)

Since Hct is available from the patient data measured prior to surgeryand V_(m) is measurable based on the method described above, the totalblood volume V_(b) can be estimated when the value of the packing ratio(η) between V_(m) and V_(c) is determined experimentally beforehand(methods and examples are presented herein, demonstrating the method fordetermining packing ratio η).

The values of the packing ratio η may be different for different bloodtypes (e.g., human versus non-human). The packing ratio value is alsolikely to be container-specific in that it may vary according to thesize and shape (aspect ratio) of the container used to collect theblood. In this regard, recall tests showed the container shape affectedthe ESR (Erythrocyte sedimentation rate). Thus, the packing ratio ηshould remain the same where the same blood type and container shape forblood measurement are used.

The packing ratio η is determined empirically by using a knownhematocrit value and a known volume of blood within a blood/salinemixture. Preferably, the value of the packing ratio η will be determinedby finding an average packing ratio value from numerous blood/salinemixture samples with different known blood concentrations. In thisregard, by running a study with blood from the same species of animal(bovine, equine, human), and a container having a defined size andshape, in the presence of the flocculant as described herein, wheredifferent known blood/saline concentration mixtures are examined, anappropriate packing ratio η may be calculated. The greater the number ofdifferent blood/saline mixtures examined, the more statisticallyreliable the average packing ratio value derived will be. As discussedabove, the blood concentration of a collected fluid in some embodimentsshould be about 50% or less blood. Additionally, an average hematocritmay be determined from a number of hematocrit values obtained from arepresentative number of subjects (animal/human). Hematocrit may becalculated using traditional capillary centrifugation methods, as knownby those of skill in the art. The average hematocrit value for a groupof animals, such as a group of humans or a group of horses, etc., willprovide a value V_(c) that can be used in the formulas and methodsdescribed herein when the blood volume is known during the particularcollection episode.

As noted, the value of the packing ratio η is preferably an averagepacking ratio value calculated from a group of blood/saline mixturesamples in a defined collection container. The actual RBC volume V_(c)and the settled RBC volume (by ordinary gravity, at room temperature),V_(m), facilitated in the presence of an RBC flocculant, will bedetermined for a number of individual samples, and an average packingratio value determined. To determine the actual RBC volume V_(c), thehematocrit of the blood added to a mixture sample is multiplied by thevolume of blood added to that sample.

Next, the packed RBC volume V_(m) may be determined for each mixturesample using conventional volumetric graduated markings on a container,as described above. That is, with the flocculant present, the V_(m) maybe determined by reading the volume of the RBC sedimentation. As part ofthe method, the same type and amount (concentration in the total fluidmixture volume) of RBC flocculant should be added to a collectioncontainer. As shown in table 2, even when the flocculant, polyDADMACconcentration was changed, the packing ratio η, remained relativelyconsistent. Thus, a range of flocculant concentrations (range offlocculant of about 0.3%, 0.4%, and 0.75% flocculant in the total liquidvolume) can be used to induce RBC sedimentation without significantlyaffecting the packing ratio. In addition, the data in Table 2demonstrates that the packing ratio is relatively insensitive to theamount of blood volume in the liquid mixture (blood/saline), when theflocculant concentration remains relatively the same.

With the actual RBC volume V_(c) and the settled, packed RBC volumeV_(m) for each mixture sample, a packing ratio value, η value, for eachsample (i.e., each different blood concentration mixture) can bedetermined. Then, an average packing ratio value may be calculated. Forbovine blood, for example, the average packing ratio calculated was 1.61(See Table 2).

To demonstrate the empirical determination of the packing ratio value,the value of the packing ratio η was determined for bovine bloodpurchased from a commercial vendor, these blood materials containingsodium citrate (for anticoagulant). Plastic canisters that were markedfor volume (ml) were used, and are shown in FIG. 4. The results areillustrated in the following table 2. Before the experiments, theaverage hematocrit Hct of bovine blood was calculated from a number ofbovine blood samples, and determined to have an average of 37.3%. Thisaverage Hct is used in the table below, and was used in the presentapproximation of blood volume in a sample. A traditional capillarycentrifugation method was used for determining individual Hct values.

TABLE 2 Experiments Hct V_(b) (ml) V_(c) (ml) V_(m) (ml) η value 30 ml20% blood concentration + 90 μl 37.3%  6 ml 2.24 3.5 1.56 flocculant(i.e., 0.3% (v/v %) concentration of polyDADMAC working solution (6 mg.)30 ml 40% blood concentration + 180 μl 37.3% 12 ml 4.48 7.5 1.67flocculant(i.e., 0.6% (v/v %) concentration of polyDADMAC workingsolution (12 mg) 50 ml 20% blood concentration + 200 μl 37.3% 10 ml 3.736.0 1.60 flocculant(i.e., 0.4% (v/v %) concentration of poyDADMACworking solution (13.3 mg) 50 ml 40% blood concentration + 200 μl 37.3%20 ml 7.46 12.0 1.61 flocculant(i.e., 0.4% (v/v %) concentration ofpolyDADMAC working solution (13.3 mg) Average 1.61 Coefficient ofVariance 2.8%

The packing ratio values that resulted from these experiments showedrelatively small variations. In these experiments, the bloodconcentration of all blood/saline mixture samples in the chart above isless than 50% since mixtures with a higher amount of blood can bediluted to provide a mixture with less than 50% blood by adding saline.After determining that the Hct is 37.3%, and the mean packing ratio ηvalue equals 1.61, the total blood volume in a blood/saline mixture canbe estimated using the following formula (derived from Equation 1):

V _(b) =V _(m)/(37.3%×1.61)=1.67×V _(m).

The number 1.67 in the above formula is the calculated value of the1/37.3% (average Hct)×1.61. (average η value from Table 2).

The following Table presents a comparison between the actual bloodvolume known to be present in each sample compared to the approximatevolume (in ml) of blood in the sample determined through the calculationof blood volume determined using the formula above and techniquesdescribed here. The results demonstrate that the formula and techniquesprovided here may be used to provide an approximation of blood volume ina liquid sample containing mammalian blood (for example, bovine blood),in the presence of a RBC flocculant (for example, a polymeric flocculantsuch as polyDADMAC). In addition, the data shows that the approximatedblood volume present in a collected mixed blood/saline liquid sampleclosely correlates with the actual blood volume in the liquid.

TABLE 3 Actual V_(b) Experiments V_(m) (ml) Approximate V_(b) (ml (ml)30 ml 20% blood 3.5  5.85 ml  6 ml 30 ml 40% blood 7.5 12.53 ml 12 ml 50ml 20% blood 6.0 10.02 ml 10 ml 50 ml 40% blood 12.0 20.04 ml 20 ml

The 20% blood noted in Table 2 is composed of 200 ml bovine blood and800 ml saline in every 1000 ml of fluid mixture.

Example 3—Fluid Collection Canister

The present example demonstrates the preparation of a particular fluidcollection container with a RBC flocculant.

Blood Containing Liquid Collection Container. The fluid collectioncontainer used in the following examples was a 1200-ml suction canister,shown in FIG. 7B. To this canister (1), a flocculant was applied,polyDADMAC, which appears as a film of dispersed flocculant particles(2) on the bottom and walls of the canister.

Flocculant—PolyDADMAC. Kemira's “Superfloc C-591” was used as the sourceof polyDADMAC. The quality or purity of this product was not consistent.Therefore, the Sigma-Aldrich version of high-molecular weight (200-350KDa) 20% polyDADMAC (Sigma Catalog #409022) was used. The resultsindicate that the Sigma version of polyDADMAC significantly improve thetesting results. However, it is anticipated that virtually any number ofdifferent sources flocculants may be used in the practice of the presentinvention, as well as in the fabrication of the herein describedflocculant containing and treated fluid collection containers.

Example 4—Optimization of PolyDADMAC as the Flocculant for a 1200 mlCollection Canister for Blood Volume Approximation in a MixtureContaining Blood

In the operating room, a biological fluid waste collection canister maybe used to collect a volume of fluid, which will include an unknownvolume of blood. The volume of blood in a canister volume of 1200 ml canvary anywhere from 10 ml to 1200 ml. The volume of blood in a collectedfluid collected may also vary depending on specific species of animal,the gender and weight of the patient, as well as the particular medicalprocedure, being performed. However, the volume of blood in a collectedfluid in a collection canister during a typical adult human surgicalprocedure is generally contains about 20% to about 50% blood.Conventional techniques for blood volume determination used in astandard hospital operating settings provide only gross estimations ofblood volume that are most times inaccurate by at least 50%-75%, and arenot available until hours after a surgical procedure has been completed.

In most cases, the amount of blood in an aspirated fluid during asurgical procedure is 50% or less. In those cases where the fluidcontains more than 50% blood, the present methods and devices may beused to accurately determine blood volume by adding saline or otherdiluent to the fluid to lower the blood concentration, so as tofacilitate the sedimentation of RBC's in the fluid in the presence of aRBC flocculant, polyDADMAC.

The present example demonstrates that a relatively constant amount ofRBC flocculant, such as polyDADMAC, may be used to achieve a relativelyaccurate estimation of blood volume is a fluid containing about 50% orless blood concentration. This is achieved by using a visual reading ofthe volume of settled RBCs in a calibrated canister containing a RBCflocculant. This volume of settled RBCs is then used in a calculation todetermine the volume of blood in the mixture. The settled RBC volumevalue alone is insufficient to accurately approximate blood volume in amixture.

A test of 40% of blood at three different volumes of a blood/salinemixture was tested. The volumes of 40% blood/saline mixtures testedwere: 200 ml, 800 ml, and 1200 ml. A Sigma-Aldrich polyDADMAC (20 wt. %in water) solution was diluted with saline to provide a working solutionof 6.67 wt. % of polyDADMAC in water. This working solution was used toprovide the appropriate amount of flocculant in this study.

Table 4 provides the optimal amount of flocculant for each volume of the40% blood/saline mixture examined. The optimal amount of flocculant wasidentified as the amount of flocculant required to provide the fastestrate of red blood sedimentation out of the mixture. The RBCs in each ofthe mixture volumes examined achieved a visually discernable level ofsedimentation in the canister, with a relatively clear fluid beingobserved above the visually discernable meniscus of settled RBCs atabout 15 minutes after the blood/saline mixture had been combined withthe flocculant at room temperature. It was observed that the rate ofsedimentation varied depending on the amount of flocculant provided inthe mixture.

From Table 4, an average of about 0.75 ml to about 1.5 ml of theflocculant working solution (about 50 mg to about 100 mg dry weightpolyDADMAC) was optimal for promoting rapid RBC sedimentation in a 100ml volume of the blood/saline mixture. For a larger 1,200 ml canisterhaving a volume of about 1200 ml, about 9 ml of the polyDADMACflocculant working solution (or about 600 mg dry weight polyDADMAC)would be provided in the bottom of the canister or on the canister wallsto promote rapid RBC sedimentation is a volume of up to 1,200 ml of theblood/saline mixture. In the following studies, about 0.75 ml to about1.5 ml of the flocculant working solution was used per 100 ml (or 0.75%v/v) of a blood/saline mixture. This concentration value is slightlylarger than the 0.4% and 0.6% used in the studies described herein whenusing the industrial grade flocculant Superflock C-591 version ofpolyDADMAC.

Based on the identified 0.75% v/v polyDADMAC concentration, a 1,200 mlcanister that can collect up to 1,200 ml of a blood-containing fluidwill need about 9 ml of the polyDADMAC working solution (or about 600 mgdry weight polyDADMAC).

TABLE 4 Optimal Amount of Need of flocculant per ⅓ diluted 100 ml ofblood saline Test blood/saline volume 20% polyDADMAC mixture 200 ml of40% blood 2-3 ml   1-1.5 ml 800 ml of 40% blood 6-8 ml 0.75~1 ml 1200 mlof 40% blood    9 ml    0.75 ml

Table 4 demonstrates the amount of flocculant that is optimal forachieving sedimentation of RBCs for different volumes of a 40%blood/saline mixture. As shown, 9 ml of the RBC flocculant polyDADMAC(about 600 mg dry weight) provided optimal RBC sedimentation in a volumeof 1200 ml of the blood saline mixture.

Example 5—Method of Preparing a Flocculant Treated Canister

The present example describes various methods in which a flocculant maybe provided and distributed within and/or on a fluid collectioncontainer, particularly a fluid containing blood. While the specificpolymeric flocculant, polyDADMAC, is used in the present example, manyother polymeric and non-polymeric flocculant may be used in the practiceof the present invention for providing the methods and devices describedherein.

Vertical Band Coating on Canister Wall. A 1,200 canister was coated witha 9 ml volume of material that contained 600 mg flocculant. The canisterwas coated in the form of 1″ wide vertical band in the canister. Thisway, only flocculant immersed by the blood/saline mixture is dissolvedor released into the mixture to cause RBC sedimentation. In other words,the vertical band of polyDADMAC coating can provide a control release offlocculant proportional to the volume of blood/saline mixture.

Two methods were used to apply the vertical band coating. One is to usea brush to provide a 1″ band directly on the canister wall. Another isto apply a flocculant coating on a 1″ wide clear tape, and then toadhere the clear tape to the canister wall. The canister has ahydrophobic surface such that it can be difficult for the flocculantsolution to stay on the wall. An ozone treatment was developed to changethe hydrophobic canister wall to hydrophilic. The flocculant adherenceusing this technique was improved.

After the coating of the vertical flocculant band, the canister wastested with bovine blood purchased from a commercial vendor. Thecanister with the flocculant band did facilitate RBC sedimentation.However, the sedimentation rate was somewhat slower. It took more than20 minutes for the RBC to settle at bottom of the canister. Generally, afaster rate of RBC sedimentation is desired. While not intending to belimited to any theory or specific mechanism of action, the slower ratemay be related at least in part to the time needed for the flocculantcoating to dissolve and spread throughout the entire blood/salinevolume. It takes more time to spread the flocculant from single-bandcoating to the entire blood/saline mixture. A thinner coating that isdistributed throughout the entire canister is expected to accelerate thedissolution and distribution of the flocculant.

Uniform Coating on Entire Canister using the Ultrasound AtomizationTechnology. An ultrasound based method was used to coat the interior ofa blood collection canister. Ultrasound atomization coating is apressureless, low-velocity (typically on the order of 3-5 inches persecond) coating that differentiates itself from spray coating.Piezoelectric transducers convert electrical input into mechanicalenergy in the form of vibrations. The high frequency sound vibrationatomizes liquid into a fine mist spray (FIG. 6). The unpressurized,low-velocity spray significantly reduces the amount of overspray sincethe drops tend to settle on the substrate, rather than bouncing off it.The mist spray pattern can be controlled and shaped precisely. Spraypatterns from as small as 0.070 inches wide to as much as 1-2 feet widecan be generated using these types of specialized spray-shapingequipment. The atomization device used in this process had a 60 kHzultrasound nozzle.

FIG. 7A illustrates the manual coating process of a canister using theultrasound atomizer. The atomizer tip moves along the canister wallstarting from the bottom of the canister gradually moving up to the topof the canister. Regions coated by misty spray turn clear wall to foggysurface, which guides the manual coating process to cover the entirecanister wall and to make the coating as uniform as possible based onvisual observation. An automatic coating process can be developed infuture to make uniform coating.

Multiple parameters were tested, using different concentrations ofpolyDADMAC, and incorporating high vaporized alcohol, like methanol andethanol, into the spray solution to accelerate the drying process. ThepolyDADMAC working solution (prepared in DI (de-ionized) water) providedan optimal misty spray to deposit the flocculant using an ultrasoundatomization device at the intensity setting of “10” (FIG. 7A). Thesyringe pump setting was 60 ml/h. About 4.5 ml of the flocculant coatingsolution was needed to cover the entire 1,200 ml canister wall.Therefore, to apply 9 ml of the flocculant solution onto the canister,two batches of the flocculant coatings were needed. A 9 ml volume of theflocculant solution contains about 600 mg polyDADMAC.

Between each coating, the canister was allowed to dry completely, eitherinside an oven for couple of hours or under room temperature for 24hours. FIG. 7B illustrates a canister applied two batches of coating inits entire wall. Use of a more powerful ultrasound atomization devicewill permit a greater concentration of the coating solution. In thismanner, a single batch of coating may be used to deliver all thepolyDADMAC contained in 4.5 ml of the solution, instead of in a 9 mlvolume, to provide the flocculant concentration described above.

The flowing protocol provides the manual steps for preparing theflocculant treated (coated) canisters:

Preparation for the Coating

1. Prepare polyDADMAC working solution by mixing one part of 20 wt. %polyDADMAC (Sigma Catalog #409022) with two parts of DI water.

2. Fill a 60 ml syringe with the polyDADMAC working solution.

3. Set the ultrasound atomization device at the intensity setting of“10”.

4. Set the syringe pump rate at 60 ml/hr., and volume deliver of 4.5 ml.

Manual Coating Process

1. Turn on the ultrasound atomization device, start moving theultrasound nozzle from the bottom of the canister applying misty sprayon the wall and bottom of the canister.

2. Gradually moving the ultrasound nozzle from bottom to the top of thecanister following the spiral path and ensuring everywhere on the wallis coated with polyDADMAC. The regions coated with the polyDADMAC mistshow misty looking as seen in the following picture.

3. Typically, the 4.5 ml diluted polyDADMAC is sufficient to provide onecoating to the entire canister.

4. Let the coated canister to dry overnight at the room temperature.

5. After the dry of the first layer of coating, a second layer isapplied to the canister in the same way as described above. A total of 9ml of diluted polyDADMAC will be applied on the canister after twolayers of coating.

6. Dry the canister again overnight, then the canister is ready to beused.

Example 6—Estimation of Packing Ratio (η) of the PolyDADMAC Coated 1200ml Canister

The polyDADMAC coated 1200 ml canister was prepared using a canisterhaving a volume of 1,200 ml, and having the dimensions of a biologicalwaste canister employed in operating rooms. A typical organization ofsuch a canister in an operating room setting is provided in FIG. 5.First, a packing ratio η associated with this coated canister wasdetermined.

Bovine blood purchased from a commercial vendor was used in the presentstudy. This blood had been refrigerated, and then warmed to 23° C. atthe time of testing. In addition, the bovine blood contained sodiumcitrate in order to prevent coagulation.

Bovine blood and saline were added to a flocculant treated canister soas to achieve a defined ratio of blood/saline. To simulate the processin which a liquid containing blood would be provided into a collectioncanister during a routine operation, for example, a 600 ml volume of ablood/saline mixture (40% blood) was delivered to a flocculant treatedcanister according to the following technique. A container A wasprepared to include 240 ml of blood, and a container B was prepared tocontain 360 ml of saline. A serological pipet was used as the aspirationprobe. The aspiration probe was connected to an inlet port (the patientport) on the canister via tubing. The canister was further connected toa vacuum line using a second port on the canister, and used to impart avacuum in the canister. Under vacuum, the pipet was placed in thecontainer including blood or the container including saline to aspiratethe respective fluid alternatively into the flocculant containingcanister (polyDADMAC) (FIG. 8). A total fluid volume of 600 ml of the40% blood was therefore provided in the canister.

After all of the blood and saline had been aspirated into the thirdcanister, the mixture was monitored to assess separation/settlement ofthe RBCs apart from the plasma and saline, in the presence of theflocculant. The volume of the RBC settlement line was recorded everyminute for 20 minutes.

The study was conducted using different volumes of the 40% blood/salinemixture solutions (200 ml, 400 ml, 800 ml and 1200 ml). Each study wasrepeated three times. FIGS. 10A-10D illustrate the change of RBCsettlement volume along the time of 20 minutes with the 40% bloodmixture. The studies were also performed three times with a 20%blood/saline mixture solution at volumes of 200, 400, 800 and 1200 ml.(FIGS. 9A-9D). In order to obtain an average packing ratio η that wouldwork for different volumes and different blood concentration mixtures ina fluid collected in this canister, two blood concentrations of 20% and40%, and four different volumes of these, 200 ml to 1200 ml, wereexamined.

With a 20% blood mixture (20% blood/80% saline), the RBCs settled veryquickly in the flocculant treated canister. A visually observablesettlement of RBCs of 30 ml (V_(m)) was reached within 10 to 15 minutesat room temperature (FIG. 11A). The average V_(m) from three repeatedexperiments and the calculated V_(ic) (based on the measured bovineblood hematocrit (35.4%), and the unknown blood volumes used in thestudy), of all four different total mixture volumes of the 20% and 40%blood preparations are listed in Table 5.

These experiments demonstrate the polyDADMAC coating facilitated therapid sedimentation of RBCs out of the blood/saline mixture at roomtemperature within 15 minutes. The sedimentation of RBCs in theseblood/saline mixtures at room temperature in the absence of a flocculantwould have required 3-6 hours.

Estimation of the Packing Ratio η. Using the data from the aboveexperiments, the packing ratio η (see Equation 1) between the settledRBC volume (V_(m)) and the actual RBC volume (V_(c)) can be determinedempirically. Table 5 illustrates the empirically determined packingratio for the 1200 ml Medi-Vac canister, which has the average value of1.20. Since the packing ratio covers a large range of volumes from 200to 1200 ml as well as a large range of blood concentrations, thevariance of the packing ratio is relatively high, above 13%. Thesevariations in packing ratio also affect the variance in blood volumeloss estimation. The following packing ratio η values were calculatedfor each of the respective blood mixtures. An average value η was thencalculated.

TABLE 5 Empirically Determined Packing Ratio: Average observed AverageVc Blood Mixtures V_(m) (ml) (ml) η (Vm/Vc) 1. 200 ml 20% blood 18 14.581.23 2. 400 ml 20% blood 30 29.16 1.03 3. 800 ml 20% blood 60 58.32 1.034. 1200 ml 20% blood 100 87.48 1.14 5. 200 ml 40% blood 30 28.01 1.07 6.400 ml 40% blood 70 56.03 1.25 7. 800 ml 40% blood 160 112.05 1.43 8.1200 ml 40% blood 233.3 168.08 1.39 Average 1.20 Coefficient of 13%Variance

In this study, the average hematocrit value of bovine blood used was35.4%, and the derived average packing ratio η was 1.2. Using theformula at Equation 1, the following formula was created to estimate thevolume of blood in a fluid collected in the flocculant containingcanister:

V _(b) =V _(m)/33.4%×1.2=2.35×V _(m)

The estimate of blood loss for each of the blood mixtures 1-8 arepresented in the following Table 6, and compared to the known amount ofblood in the sample.

TABLE 6 V_(m) Estimated blood Actual blood Blood Mixtures (ml) volume(ml) volume (ml) 1. 200 ml of 20% blood 18 42.30 40 2. 400 ml of 20%blood 30 70.50 80 3. 800 ml of 20% blood 60 141.0 160 4. 1200 ml of 20%blood 100 235.0 240 5. 200 ml of 40% blood 30 70.5 80 6. 400 ml of 40%blood 70 164.5 160 7. 800 ml of 40% blood 160 376.03 320 8. 1200 ml of40% blood 233.3 548.3 480

As demonstrated in the table above, the amount of blood loss calculatedusing the present formula was correlated with the actual amount of bloodin the fluid. The present methods and devices therefore are demonstratedto provide a contemporaneous visual indicator tool of blood volume lossfor the physician/health care professional in a surgical setting, whichis more accurate than conventional approaches (saline bag use assessmentand/or post-surgery estimation from total patient fluid collection).

Based on an observed volume (in ml) of settled RBCs in a graduatedcanister in the presence of a flocculant, without the requirement of anyelectrical, temperature, or other material manipulating procedure, aBlood Indicator Panel was devised using the above formula, that providesan immediate visual tool for total blood volume approximation in acollected fluid.

Example 7—Creation of a Blood Volume Indicator Panel for Blood VolumeAssessment in a Biological Fluid Receptacle

For a user to easily estimate blood volume via a visual inspection ofsettled RBCs in a receptacle (such as a flocculant containing canister),a blood volume indicator panel with calibrated markings is provided fora fluid collection receptacle, and will indicate a total blood volumeapproximation in a collected fluid, from the level of settled RBCs inthe presence of a flocculant in the collection receptacle. It isenvisioned that these collection receptacles may or may not includeconventional volumetric measures.

To create the graduated marking for the blood volume indicator panel ofthe present receptacles (canisters, etc.), the following Equation 2 isused:

V _(m) =V _(b) ×Hct×η  2)

The Equation 2 will employ an average Hct calculated from a number ofanimals/human from the same species, and of the same approximate age andgender. For example, for an adult human male, the average Hct is about45%.

In this study, a blood indicator panel for a large mammal was createdusing the formula:

V _(m)=0.43×V _(b)  Equation 3:

The formula will use and average Hct in bovine blood of 35.4%, and anaverage packing ratio η=1.20, as calculated for bovine blood (Table 5).

A 50 ml estimated blood volume mark is provided on the blood volumeindicator panel, which corresponds to a visually discernable settled RBCvolume of about 21.5 ml. using the Equation 3 (See Table 7). A 100 mlestimated blood volume calibrated mark can be generated on the bloodvolume indicator panel that corresponds to about the 43.02 ml of thesettled RBC volume line of the receptacle, and so forth.

The graduated markings of the blood volume indicator panel provide aseries of visually identifiable markings that do not relate to a measureof the volume of material in the canister, but instead to anapproximation of the blood volume in the fluid collected in thecanister. An illustration of a typical blood indicator panel (BIP), isprovided in FIG. 15 (See Left Panel 6, calibrated markings at 50 ml, 100ml, 200 ml, 400 ml, 600 ml). Inclusion of a Blood Indicator Panel on aconventional collection vessel having standard volumetric markings (SeeFIG. 16, Right Panel 1), will provide an immediately visual estimationof blood volume in a collected fluid, without the necessity ofperforming mathematical calculations or other manipulations of collectedor sedimented materials. As shown, the blood volumes identified with thecalibrated markings of the BPI do not coincide with the conventionalvolume of settled RBCs in the fluid. Instead, the volume of settled RBCsis used as part of a calculation together with hematocrit and thedefined aspect ratio to provide an approximation of blood volume. In theabsence of a flocculant, the volume of blood in a liquid would not bepossible to approximate within less than about 3-6 hours because, amongother things, RBCs do not begin to settle until well after 3-6 hours. Inaddition, the presence of flocculant, alone, while facilitating rapidRBC sedimentation, does not immediately approximate the amount of bloodin a liquid. As demonstrated in the present results, the volume ofsettled RBCs in a liquid was less than about 50% of the actual bloodvolume known to be contained in a test fluid containing a known amountof blood. The volume of settled RBCs in the presence of flocculant hasto be further corrected to accommodate blood average hematocrit andpacking ratio, to provide an approximate blood volume in a liquid.

The BIP Panel is created based on the derivation of an averagehematocrit (Hct), for example, the average Hct for human, bovine,equine, etc. To correct for Hct differences in individualpatients/animals, such as differences in individual Hct due to gender,species, age, etc., the approximate blood volume value indicated on theBIP may be adjusted by a factor that corrects for significantly higheror lower individual hematocrit values. For example, if the measured Hctvalue from a patient is lower, for example, 80% of the typical Hct of anadult human male, then the blood volume indicator value on the panelobserved for that patient will be divided by 80%, so as to provide aneven closer approximation of the estimated blood volume in thereceptacle. More particularly, if the blood volume indicator value onthe panel is 50 ml according to the graduated markings on the indicatorpanel, the actual blood volume in the biological material collected fromthis patient would be calculated to be 62.5 ml where the patient's Hctis 80% of the typical Hct value. Similarly, if the measured Hct value ofa patient is higher, for example 110% of a typical adult male hematocritvalue, then the blood volume indicator value on the blood volumeindicator panel would be divided by 110%, which will yield a lower bloodvolume. For example, if the blood volume indicator value on the bloodvolume indicator panel is 100 ml, then the actual blood volume loss forthe patient would be calculated to be 90.9 ml, so as to correct for thepatient's higher than average Hct. (e.g., 10% higher). FIG. 8illustrates a 1200 ml canister with a blood volume indicator panel.

The particular Blood Indicator Panel as placed on a collection canisteris shown in FIG. 12. The BIP was created to provide a blood volumeestimation in a volume of a 20% blood/saline mixture or in a 40%blood/saline mixture using bovine blood. The Blood Indicator Panel mayalso be useful for assessing the volume of human blood in a liquidsample. This is because both bovines and humans are mammals, and bloodfrom bovines and humans share many characteristics, including similaraverage hematocrit.

Table 7. Example of the Blood Indicator Panel markings to be used on a1200 ml canister. These calibrated blood volume markings correspond toan estimate of the volume of blood contained in a fluid sample, comparedin the table to the corresponding sedimented RBC volume (indicated bythe value V_(m) in the table). (Bovine blood with sodium citrate)

Graduated Volume Marking of Calibrated Approximation Sedimented RBCs(V_(m)) of Blood Volume (V_(b)) (about 20% blood to 50% Blood containingBIP Fluids)  50 ml 21.5 ml 100 ml 43.0 ml 200 ml 86.0 ml 400 ml 172.0ml  600 ml 258.0 ml 

This study successfully developed the prototype for a BIP using a RBCflocculant, polyDADMAC coated biological fluid collection canister(1200-ml). This size canister is used in operating rooms for humanpatients, adult and pediatric. The evaluation of the prototype usingbovine blood has shown a quick sedimentation of RBCs in the presence ofthis exemplary RBC flocculant, and the achievement of stable RBCsedimentations within 20 minutes. Calibrated markings on the speciallydesigned BIP were designed for this collection canister, and may be usedto provide a visual estimation of total blood volume in a collectedmammalian liquid. If the patient's measured hematocrit is different fromthe typical hematocrit used to create the calibrated BIP markings, thecalibrated blood volume can be corrected to accommodate the percentblood hematocrit to the individual blood volume amount.

The following provides the average hematocrit for an adult man and foran adult woman: Normal Hct Values: Men—42-52% (Average Hct, 47);Women—37-47% (Average Hct, 42).

Example 8—Creation of 100 ml Canister with Flocculant

A 100 ml canister was prepared, to contain about 50 mg flocculant. Inthis example, the RBC flocculant used was polyDADMAC. The flocculant wasprovided in a volume of the polyDADMAC working solution describedherein.

In a typical operating room setting, smaller volumes of fluid containingblood and other materials (tissue, urine, non-blood fluid, etc.) will beaspirated from a surgical field. The aspiration of these fluids resultsin an undetermined loss of blood from the patient. A smaller containermay be prepared according to the present invention to accommodate theestimation of blood loss in these small, sometimes critical, volumes ofcollected fluid. Therefore, these 100 ml receptacles containing a RBCflocculant, such as polyDADMAC, are provided and are especially usefulfor determining blood volume in small amounts of collected fluid. Thesedevices may be used, for example, in pediatric applications (infant) aswell as in low volume critical fluid collection procedures.

The aspect ratio of the 100 ml collection device was calculated to be0.96. The small 100 ml container with the RBC flocculant was used in thestudy described in Example 9.

Example 9—Fresh Blood Loss Estimation

The present example is provided to demonstrate the utility of themethods and devices for use for estimating blood loss in a fluidcontaining mammalian fresh blood (no anti-coagulants). In this example,a non-blood material present in the fluid was saline. The presentexample examines a technique for estimating blood loss using fresh,never refrigerated, volume of mammalian blood. Further, the blood didnot contain calcium citrate, or any other anti-coagulants. In thepresent study, the fresh blood specimen was obtained from an adulthorse. Thus, the devices and methods are especially useful in theapproximation of blood loss in mammals, including humans and veterinaryanimals (horses, dogs, cats, cows, bulls, sheep, pigs, etc.).

In this example, blood was collected from a live, adult horse(approximately 12 years old, weight 1,200 pounds), having no knownclinical pathologies, and not on any known medications. The animal wasbeing treated for a lame foot, and was being given a nerve block tomanage pain. Unlike blood collected from a commercial vendor, whichcontains an anticoagulant such as sodium citrate (used in the priorexamples), no anticoagulants or other drugs were present in the bloodcollected from the horse used in this study.

A total of twelve (12) canisters having a total volume capacity of 100ml were used in the present study. The canisters were marked withdemarcations along the side of the canisters at 50 ml and 100 mlincrements. The aspect/ratio of the 100 ml container, D:H (Diameter vs.Height) was calculated to be about 0.96. Comparatively, the aspect ratioof the 1200 ml canister is about 0.61. Typically, the larger the aspectratio of the collection device, the more quickly RBCs contained withinany blood in the collected liquid will settle out in the collectiondevice. Therefore, the rate of RBC sedimentation in the 100 mlcollection device was expected to be more rapid compared to thesedimentation rate of RBCs in a 1200 ml canister, in the presence of theRBC flocculant, under similar conditions.

The 100 ml dry canisters received 50 mg of the RBC flocculant,polyDADMAC, research grade (Sigma Catalog #409022). (See Example 7).Other RBC flocculants, as well as industrial grade versions of theseflocculants, including PEI, PAM and others, may be expected to be usefulin the present methods and devices.

The amounts of fresh equine blood and saline indicated in Table 8 werethen added to each of the canisters, and RBC sedimentation volume wasrecorded every minute for 20 minutes:

TABLE 8 Fresh Equine Blood Study: Rate of Sedimentation of RBC RBCSedimentation/ml (V_(m)) Blood/Saline 1 min 2 min 4 min 5 min 10 min 15min 20 min With Flocculant 1. 10% Blood 10 ml/90 ml 50 40 30 10 ml 10 ml10 ml 10 ml 2. 20% Blood 20 ml/80 ml 60 50 50 20 20 20 20 3. 30% Blood30 ml/70 ml 90 90 60 30 30 30 30 4. 40% Blood 40 ml/60 ml 100 100 80 4545 30 40 5. 50% Blood 50 ml/50 ml 100 100 75 45 45 45 45 6. 65% Blood 65ml/35 ml 100 100 75 60 65 65 60 Controls: Without Flocculant 7. 10%Blood 10 ml/90 ml ND ND ND ND ND ND ND 8. 20% Blood 20 ml/80 ml ND ND NDND ND ND ND 9. 30% Blood 30 ml/70 ml ND ND ND ND ND ND ND 10. 40% Blood 40 ml/60 ml ND ND ND ND ND ND ND 11. 50% Blood  50 ml/50 ml ND ND ND NDND ND ND 12. 65% Blood  65 ml/35 ml ND ND ND ND ND ND ND * ND =Non-detectable by visual inspection.

Blood from an adult horse was drawn, and a volume of the fresh blood atbody temperature was added to each of the canisters in the amountsindicated above. The top on each of the canisters was then put in place,the contents mixed so as to assure proper mixture of saline, blood andflocculant. Each canister was allowed to sit undisturbed at roomtemperature, and observed. The time at which sedimentation of RBCs wasobserved was recorded at 1 minute intervals up to 30 minutes. FIG. 18presents the RBC sedimentation rates of the various mixtures.Essentially no visually detectable RBC sedimentation was observed withthe blood mixtures in the absence of flocculant. In contrast, RBCsedimentation was rapidly observed in all blood mixtures containingflocculant within 5 minutes at room temperature.

Table 9 presents the hematocrit of various large animals. The values formean corpuscular hemoglobil (MCH), mean corpuscular hemoglobinconcentration (MCHC), mean corpuscular volume (MCV), and packed cellvolume (PCV), and may be employed in providing an appropriate customizedBlood Indicator Panel and blood volume approximation method as describedin the present disclosure.

TABLE 9 Normal Values for Erythron Data in Ruminants and the HorseCattle Sheep Goats Horses PCV (%) 24-46 27-45 22-38 32-53 Erythrocytes(× 10⁶/L)  5-10  9-15  8-18  6.7-12.9 Hemoglobin (g/dl)  8-15  9-15 8-12 11-19 MCV (fl) 40-60 28-40 16-25   37-58.5 MCH (pg) 11-17  8-125.2-8   12.3-19.7 MCHC* (g/d1) 30-36 31-34 30-36   31-38.6 Reticulocytes0 <0.5% 0 0   Erythrocyte diameter (m) 4-8 3.2-6   2.5-3.9 5-6Erythrocyte fragility (percent NaCl) Minimum 0.52-0.66 0.58-0.76 0.740.54 (beginning hemolysis) Maximum 0.44-0.52 0.40-0.55 0.44 0.34(complete hemolysis) Erythrocyte 0   1-2.5 0 50-60 sedimentation rate(mm/1 hour) Erythrocyte life span 160  140-150 125 140-150 (days) (MCH,Mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobinconcentration; MCV, mean corpuscular volume; PCV, packed cell volume.)

TABLE 10 Normal Values for Leukogram Data (Adult Aminals) BiologicalComponent Cattle Sheep Goats Horses White blood cells (×10³/μl)  4-12 4-12  4-13  5.4-14.3 Neutrophils (×1.0³/μl) 0.6-4   0.7-6   1.2-7.22.3-8.6 Bands (×10³/μl)   0-0.12 Rare Rare 0-1 Lymphocytes (×10³/μl)2.5-7.5 2-9 2-9 1.5-7.7 Monocytes (×10³/μl) 0.025-0.84    0-0.75  0-0.55 0-1 Eosinophils (×10³/μl)   0-2.4 0-1 0.05-0.65 0-1 Basophils(×10³/μl)   0-0.2   0-0.3   0-0.12   0-0.29 Neutrophil/lymphocyte (N:L)ratio 0.3-0.6 0.3-0.7 0.6-3.6 0.8-2.8

TABLE 11 Normal Values for Hemostatic Data in Ruminants and the HorseBlood Component Cattle Sheep Goats Horses Platelet count (×10⁻³/L)100-800 250-750 300-600 100-600 Fibrinogen (mg/dl) 200-500 100-500100-400 200-400 Prothrombin time(s) 22-55 —*  9.5-12.5 7-9 Activatedpartial 44-64 — 28-52 37-54 thromboplastin time(s) Fibrin/fibrinogendegradation <8 <8 — <32 products (μg/ml) (Modified from Duncan J R etal: Veterinary laboratory medicine, Ed 2, Ames, Iowa, 1986, Iowa StateUniversity Press; and Kaneko J J: Clinical biochemistry of domesticanimals, Ed 3, New York, 1980.)

Example 10—Blood Volume Estimation in a Fluid Using a Canister withFresh Equine Blood (No Anti-Coagulant) in the Presence of a Flocculant

The present example was performed with fresh blood drawn from an adulthorse (12 years old, about 1,200 pounds). The horse blood did notcontain any anticoagulant. The hematocrit of horse blood is between 32%and 53%. The horse blood was used immediately after having been drawn,and was at body temperature (about 101° F., 38.3° C.) at the time it wascombined with saline in the presence of flocculant, polyDADMAC (600 mg).

A 1,200 ml canister that had been coated by spray application of about 9mls of polyDADMAC working solution as the flocculant was used. Thus, the1,200 ml canister was coated evenly with a total of about 600 mg dryweight polyDADMAC as the flocculant. Because the flocculant was evenlydistributed along the walls of the canister, the amount of flocculant isreleased in proportion to the volume of fluid provided in the treatedcanister.

The following volumes of settled RBCs were recorded over time.

A volume of 400 ml fresh horse blood (not refrigerated, bodytemperature, no anti-coagulants), was placed in the treated canister. Aknown volume of 250 ml saline was added to the canister. The totalvolume was 650 ml of fluid in the canister. This provided a 61.5% bloodsolution. The volume of settled RBCs (gravity only, no centrifugation),V_(m), was recorded immediately upon mixing, up to a period of 30minutes.

After being allowed to settle undisturbed for 30 minutes, the canisterwas manually agitated, and then allowed to sit at room temperatureagain. The agitated blood containing canister was again observed forevidence of settlement of RBCs at room temperature.

TABLE 12 Time (min) RBC (settled RBC) 0 sec 600 ml 20 sec 600 ml 30 sec575 ml 1 min 500 ml 1.5 min 500 ml 2.0 min 450 ml 2.5 min 440 ml 3.0 min410 ml 4.0 min 400 ml 5 min 395 ml 6 min 360 ml 7 min 360 ml 8 min 350ml 9 min 350 ml 10 min 350 ml 11 min 340 ml 12 min 330 ml 13 min 320 ml14 min 310 ml 15 min 310 ml 16 min 305 ml 17 min 305 ml 18 min 305 ml 19min 300 ml 20 min 300 ml 21 min 300 ml 22 min 300 ml 23 min 295 ml 24min 295 ml 25 min 295 ml 30 min 295 ml

At the end of the 30 minute observation period, the canister wasmanually shaken, and allowed to sit. The re-sedimentation of RBCsoccurred, and was observed every minute for 30 minutes, and resulted inthe observation of settled RBC levels as indicated in Table 14.

TABLE 14 Time RBC (settled RBC)  0  0 ml   1 min  0 ml   2.5  0 ml 3.0min 350 ml 4.0 min 300 ml 5.0 min 300 ml   8 min 300 ml 10 300 ml 20 300ml 30 300 ml 40 300 ml  50 min 250 ml

From this study it is demonstrated that the settled volume of RBC's in asolution containing whole fresh blood remains relatively stable up toabout 30-40 minutes at room temperature. With agitation, it appears thatRBCs in the fresh blood sample again settled to provide a discernableRBC sedimentation volumetric line, V_(m), very quickly (3 to 4 minutesverses 16-19 minutes, settled RBC volume about 250 ml to 300 ml.). Theactual known volume of fresh blood present in the fluid was 400 ml.

The total approximation of blood volume may be calculated by the formulausing the average hematocrit of the type of animal (42.5%, horse averageHct), the V_(m) (observable settled RBC volume in ml), and a new packedratio value (n) determined for horse blood via experimentation withmultiple equine blood sample assessments of settled RBC volume (V_(m))and Hct information, a visual Blood Indicator Panel may be created andprovided along the vertical axis of a collection canister for use withlarge animals, such as horses. This would provide an immediatelyvisually discernable approximation of blood volume in a biological fluidcontaining horse blood. The development of a Blood Indicator Panel for atreated canister or other receptacle may be used to provide a visualindicator of equine blood volume loss, and especially for assessing amore exact equine blood loss, than is presently available. An equineBlood Indicator Panel may be developed employing the information andresults presented here, by one of ordinary skill in the veterinary arts,without more than ordinary and routine experimental optimization trialand error.

Example 11—Human Pediatric Applications for Use in Estimating Blood Loss

The present example is provided to provide canisters and methods forefficient measurement of blood loss in a pediatric patient. As used inthe present example, a pediatric patient is defined as an individual upto 12 years of age having a body weight of up to 70 to 80 pounds.

A person's total blood volume (TBV) is related to body weight. The TBVof a child is around 75-80 ml/kg and is higher in the neonatal period(from 85 ml/kg it rises to a peak of 105 ml/kg by the end of the firstmonth and then drops progressively over ensuing months). Thus, the TBVof a 3.5-kg 2-week-old will be about 350 ml while that of a 10-kg15-month-old will be about 800 ml.

Because of the much reduced total volume of blood in a pediatricpatient, it is especially important to provide a blood collection andblood loss estimation system and device that are designed for estimatingblood loss accurately from a smaller volume of blood collected from apediatric patient. The specifically designed pediatric blood lossestimation devices of the present invention are therefore crafted with acontainer having the herein described flocculant and canisterdemarcations with a total volume capacity of less than 1000 ml, such asabout 500 ml or even about 250 ml, in the case of an infant or neonate.

A large acute loss of blood volume in a pediatric patient may compromisethe circulation, and therefore blood loss should be carefully monitoredso as to be able to detect a volume of blood loss of about 12% of theTBV (around 10 ml/kg) of the specific pediatric patient, assuming thechild is in a stable condition and has a normal blood hemoglobin (Hb)level at the beginning of a procedure.

By way of example, a suitable pediatric blood loss collection devicewould, in some embodiments, have a capacity of 250 mls. The canisterwould preferably provide an appropriate aspect ratio of D:H for atypical pediatric blood loss volume. The D (diameter) of the devicewould typically be between 2 and 3 inches, and have an H (height) ofabout 2 inches to about 3 inches. With these smaller dimensions, acollected blood loss volume would provide a reasonably rapid yetmonitorable sedimentation rate of RBCs so as to alert an attendingphysician if an amount of blood loss has reached a volume wheretransfusion to the pediatric patient is in order. It would be preferredthat a sedimentation rate would be achieved that provides for RBCsedimentation within 15 minutes of blood collected in the canister.

In some embodiments, the 250 ml container has a conical shape (FIG. 14).The flocculant will be provided to the container either at the time ofthe surgical intervention event, or may be provided as a pretreatment tothe canister (such as by a spray coating).

The amount of flocculant to be added to a 250 ml collection device wouldbe about 50 mg to about 150 mg, or about 125 mg, or an amount sufficientto achieve at least a 0.3%, 0.4% or 0.75% of total volume of thesolution.

For sake of description, the following average total blood volume in apediatric group of patients may be used in calculating when a 12% orgreater blood loss has occurred. An average hematocrit value may also becalculated for the class/group (premature neonate, full term neonate,infant) of pediatric patients, and a marking provided alongside one axisof the canister, of average hematocrit values for these patient groups,so as to provide a ready visual reference for the attending physician oranesthesiologist to refer to and compare as against the hematocritobtained for the patient undergoing the procedure:

-   -   Premature Neonates 95 ml/kg    -   Full Term Neonates 85 ml/kg    -   Infants 80 ml/kg

The total approximation of blood volume may be calculated by the formulausing the average hematocrit of for a human child of a particular weightrange and/or age, or for a human adult male or adult female, the V_(m)(observable settled RBC volume in ml), and the packed ratio value (n)determined for human blood. With multiple human blood sample assessmentsof settled RBC volume (V_(m)) and Hct information, a visual blood volumeindicator panel to be located along the vertical axis of the collectioncanister may be prepared for the human, and especially for a pediatrichuman model. This would provide an immediately visually discernableapproximation of blood volume in volumes less than about 250 ml,contained in a biological fluid containing human blood. The developmentof a vertical canister or other receptacle having a Blood IndicatorPanel for human blood volume assessment in a liquid, and especially forassessing small volumes of human blood loss, may be developed by one ofordinary skill in the art given the teachings provided herein, withoutmore than a routine and ordinary amount of trial and error.

Example 12—Collapsible Treated Containers for Blood Loss Collection

The present example presents a collapsible plastic-like container (bag)that may be used to collect biological fluid loss, and used to estimateblood loss. Such a collection device is envisioned to be especiallyuseful in combat situations, or any other situation where space formedical equipment is limited.

It is envisioned that the plastic bag containers will include an amountof an RBC flocculant suitable for providing the RBC sedimentation andthe blood loss estimate features described herein. In some aspects, thebag could be placed within a supporting container, such as a box,canister, or other structure. The bag may also include a number ofmarkings along the vertical axis of the bag, corresponding to volumetricmeasures (such as milliliters).

In some aspects, a clear plastic bag having a volume capacity of about1,000 ml containing between about 300 milligrams and about 4,700milligrams of a flocculant, such as polyDADMAC, will be placed in thebag. The bag will include, in some embodiments, calibrated demarcationsat a 50 ml, 100 ml, 200 ml, 250 ml, 400 ml, 500 ml, 600 ml, 750 ml and 1liter marker. The bag may also include a BIP, such as in the form of anadhesive strip, which may be placed on the bag and used to provide avisually discernable indicator of approximated blood volume in a liquidbased on the settled RBC level in the collection bag/container. Anexemplary rendition of this embodiment is provided at FIG. 13. An insertbag having the RBC flocculant and calibrated BIP for human blooddesigned for a 1200 ml collection canister, such as the canister shownat FIG. 11A, is also provided. In such embodiments, the canister itselfneed not be treated with RBC flocculent, and instead, the insert bagwill contain the RBC flocculent. The insert bag may also optionally alsoinclude a calibrated BIP for human blood.

Example 13—Blood Loss Collection Kit

The flocculant containing canisters (1.2 ml, 500 ml., 250 ml., 10 ml),that includes a blood volume indicator panel, may be provided togetheras a kit with a length of aspiration tubing and a second length oftubing suitable for adding saline into a canister and/or collapsibleenvelope.

An instructional insert may be provided as part of the kit for the enduser.

Example 14—Stability Testing High Temperature Ageing

This example demonstrates the stability of the RBC flocculant polyDADMACand retained activity for providing RBC coalescence (flocculation) in afluid containing blood, after exposure of the polyDADMAC coated canisterto high temperatures.

In the present study, the flocculant used was polyDADMAC provided as acoating on a canister for collecting a material, such as a biologicalliquid material collected during a surgical procedure that will containa component of blood. The coated canisters were incubated at 55° C. for6 weeks (equivalent to one-year shelf life at room temperature). Thecoated canisters, after high temperature aging, were then compared inthe function test with the coated canister without going through thehigh temperature aging test.

Materials:

-   -   1) Control Group—four Canisters (Cardinal Health), coated with        600 mg of polyDADMAC (FIG. 1), allowed to dry overnight at room        temperature (≈22° C.).    -   2) Experimental Group—four Canisters (Cardinal Health), coated        with 600 mg of polyDADMAC, allowed to dry overnight at room        temperature (≈22° C.). Then the canisters were incubated in a        convection oven set at 55° C. for six weeks.    -   3) Bovine whole blood (Innovative Research Lot #24301), stored        in refrigerator and warm up to room temperature before the        experiment.    -   4) Isotonic Saline (Thermo Scientific, Lot #994448) at room        temperature.

Methods:

-   -   1) Four canisters were chosen randomly from the experimental and        control lots, respectively.    -   2) Bovine whole blood purchased from a commercial vender (sodium        citrate containing), was mixed with isotonic saline at room        temperature to the concentrations of 20% and 40% v/v of blood,        respectively. The 20% blood mixture had a total volume of 1000        ml and the 40% blood mixture had a total volume of 500 ml.    -   3) Canisters were tested two at a time: one experimental vs. one        control. Mixed blood and saline solutions were introduced into        the canister via vacuum aspiration.    -   4) The settlement of the red blood volume was recorded every        minute for 20 minutes, according to the existing graduations on        the canister.    -   5) After 20 minutes, images were taken comparing the two groups.    -   6) Data was charted as a function of RBC settlement volume vs.        time for comparison.

Results: FIGS. 16 A and 16B compares the settlement of RBCs after bloodsaline mixtures were introduced to the control and experimental(heat-treated) canisters. The tests were performed using 1000 ml of 20%blood with saline mixture (FIG. 16A), and 500 ml of 40% blood withsaline mixture (FIG. 16B). Both contain 200 ml of bovine blood. Eachtest was repeated once. The data illustrates a closely overlay of thevolume change curves of RBC settlement in the 20% blood test. All RBCvolume settlements were stabilized around 125 ml around 15 minutes afterthe mixtures were introduced the into the control and experimentalcanisters. In the 40% blood test, although the volume settlement of oneexperiment was lagged, all the volume settlements were stabilized around125 ml after 15 minutes. FIG. 17 illustrates the pictures of the settledRBCs in both control and experimental canisters. FIG. 17A (Panels I andII) and FIG. 17B (Panels I and II) compares the settlement of RBCs afterblood and saline mixtures were introduced to the control andexperimental (heat-treated) canisters. The tests were performed using1000 ml of 20% bovine blood with saline mixture (FIG. 17A), and 500 mlof 40% blood with saline mixture (FIG. 17B). Both fluids were known tocontain 200 ml of bovine blood. Each test was repeated once.

The data illustrates a closely overlay of the volume change curves ofRBC settlement in the 20% blood test. All RBC volume settlement levelswere stabilized around 125 ml at about 15 minutes after the mixtureswere introduced into the control (FIG. 17A) and experimental (FIG. 17B)canisters.

In the 40% blood test, although the volume settlement of one experimentlagged, the volume of settled RBCs was stabilized at about the 125 mlvolumetric mark after 15 minutes. FIGS. 17A and 17B illustrate thesettled RBC volumes in the control (17A) and experimental (17B)canisters.

The studies demonstrated no discernable difference in the functionbetween the control and experimental canisters after heat treatment. ThepolyDADMAC coated canisters, after six-week of aging test under 55° C.,show no functional degradation of the polyDADMAC or decrease ineffectiveness for facilitating floculation of RBCs in a liquid. Thefloculent coated cannisters (polyDADMAC coated canisters) are expectedto have at least a one-year shelf life without loss of the flocculantactivity to provide blood volume estimation in a fluid stored at roomtemperature.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the methods for prediction of the selectedmodifications that may be made to a biomolecule of interest, and are notintended to limit the scope of what the inventors regard as the scope ofthe disclosure. Modifications of the above-described modes for carryingout the disclosure can be used by persons of skill in the art, and areintended to be within the scope of the following claims.

It is to be understood that the disclosure is not limited to particularmethods or systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

We claim:
 1. A visual method for approximating blood volume in a fluidobtained from a subject comprising: receiving a volume of a fluid from asubject having a known hematocrit into a container comprising an RBCflocculant; visually determining a volume of settled RBCs in thecontainer for a period of time sufficient to permit stable sedimentationof RBCs; and determining a volume of blood in the fluid, wherein thestable sedimentation level of RBCs is calibrated for a RBC packing ratiofor the container and an average hematocrit to provide the visualapproximation of blood volume in the fluid.
 2. The visual method ofclaim 1 wherein the RBC flocculant is polyDADMAC in an amount of about0.3% to about 0.75% in a container having a volume of about 1200 ml. 3.The visual method of claim 1 wherein the subject is a human.
 4. Thevisual method of claim 1 wherein subject is a veterinary animal.
 5. Thevisual method of claim 1 wherein the volume of blood in the biologicalfluid is a volume, V_(b), calculated according to the formula:V _(m) =V _(b) ×Hct×η.
 6. A container comprising at least two inletports, an RBC flocculant, and a volume capacity of about 100 ml, about250 ml, about 500 ml, about 1,000 ml, about 1,200 ml or about 2,000 ml.7. The container of claim 6 wherein the RBC flocculant is polyDADMAC inan amount of about 0.3% to about 0.75%, and the volume capacity of thecontainer is about 1,200 ml.
 8. The container of claim 6 comprising aseries of calibrated blood volume markings, wherein said markings do notcorrespond to volumetric liquid measurements of the container.
 9. Thecontainer of claim 6 comprising a cap, said cap having at least oneinlet port suitable for receiving an aspiration tube, and at a secondport suitable for imparting a vacuum suction in said container.
 10. Acollapsible fluid collection container comprising an RBC flocculant, aseries of calibrated blood volume markings, at least two ports, amounting loop, and a volume capacity of about 250 ml, about 500 ml.,about 750 ml., about 1,200 ml., or about 2,500 ml.
 11. The collapsiblefluid collection container of claim 10 wherein the RBC flocculantcomprises polyDADMAC.
 12. A blood indicator panel for use inapproximating blood volume in a fluid, said panel comprising a series ofcalibrated markings corresponding to an approximated blood volume in aliquid comprising mammalian blood.
 13. The blood indicator panel ofclaim 12 where said calibrated markings are calculated from a volumemeasure of settled RBCs within a collection device in the presence of anRBC flocculant, a defined RBC packing ratio of a fluid collectiondevice, and an average hematocrit value of an animal of interest. 14.The blood indicator panel of claim 13 wherein the RBC flocculantcomprises polyDADMAC.
 15. The blood indicator panel of claim 13 whereinthe series of calibrated markings are not indicative of a metricvolumetric measure of a liquid in the collection device.
 16. The bloodindicator panel of claim 13 wherein the series of calibrated markingsprovides an approximation of blood volume in a fluid material obtainedfrom an adult human collected in the collection device, said bloodindicator panel being calibrated to coincide with an average hematocritof blood from the adult human and a RBC packing ratio determined for acollection device having a volume of about 1200 ml.
 17. The bloodindicator panel of claim 12 where the animal is a horse, a human or acow.
 18. The collapsible fluid collection container of claim 10comprising a bag having volume of about 1200 ml, and being suitable forplacement in a 1200 ml collection canister.
 19. The collapsible fluidcollection container of claim 18 wherein the bag comprises a bloodindicator panel, the blood indicator panel comprising a series ofmarkings calibrated to provide a visual assessment of blood volume in aliquid contained in the bag.